DDRGK1介导类泛素化修饰RPS3调控椎间盘退变的作用和机制研究

With the acceleration of aging, spinal degeneration seriously affects the quality of life of the population and increases the burden of medical treatment. In our previous study: (1) High throughput screening of differential proteins in degenerative nucleus pulposus cells shows DDRGK1 expression in degenerative nucleus pulposus cells. (2) The expression of DDRGK1 in degenerative intervertebral disc tissue and degeneration nucleus pulposus cells decreased. (3) DDRGK1 inhibits proliferation in the silent nucleus pulposus cells and increases the catabolism of extracellular matrix. (4) Overexpression of DDRGK1 reverses the degeneration of nucleus pulposus cells. (5) CoIP combined mass spectrometry analysis showed that DDRGK1 is combined with RPS3 and mediates ufmylation of RPS3. According to this hypothesis, the decreassing expression of DDRGK1 in degenerative nucleus pulposus cells induces ufmylation of RPS3 decreasing, directly affecting downstream p65-p50, p53 and E2F1 which inhibits proliferation, promoting apoptosis, exacerbating extracellular matrix catabolism, leading to intervertebral disc degeneration. According to this phenomenon, we have constructed the nucleus pulposus specific DDRGK1 conditional knockout mice and we will clear the function and mechanism of DDRGK1 in maintenance of intervertebral disc homeostasis from mouse phenotype, cell phenotype, the ufmylation of RPS3 by DDRGK1, its downstream molecular mechanism and target therapy for intervertebral disc degeneration model, to provide new ideas for clinical diagnosis and treatment of intervertebral disc degeneration.

随着老龄化加速，脊柱退变严重影响人群生活质量，增加医疗负担。我们前期研究：（1）高通量筛选退变髓核细胞中差异蛋白，发现退变髓核细胞中DDRGK1表达下调；（2）退变椎间盘组织和退变髓核细胞中DDRGK1表达下降；（3）沉默髓核细胞内DDRGK1抑制增殖，加剧细胞外基质分解代谢；（4）过表达DDRGK1逆转髓核细胞退变 ；(5)CoIP联合质谱分析发现DDRGK1与RPS3结合以及RPS3能被类泛素化。据此假说“退变髓核细胞的DDRGK1表达下降，减少RPS3类泛素化，直接影响下游p65-p50、p53及E2F1抑制增殖，促凋亡，加剧细胞外基质分解代谢，导致椎间盘退变”。为此，我们已经构建髓核DDRGK1条件性敲除小鼠，将从小鼠表型、细胞表型、DDRGK1调控RPS3类泛素化及其下游分子机制，椎间盘退变模型靶向治疗等层面阐述DDRGK1维持椎间盘稳态的功能和机制，为临床诊断和治疗提供新方向。

椎间盘退变表现为腰背痛碍，严重影响老年人生活质量。我们从小鼠表型、细胞表型、DDRGK1调控RPS3、IRE1a、QSOX1类泛素化的分子机制，针对该机制挽救小鼠表型等层面阐述DDRGK1/UFM1维持髓核细胞的稳态。我们研究发现：1）退变髓核细胞内DDRGK1蛋白水平下调；2）Crispr/Cas9敲除DDRGK1的小鼠髓核及软骨退变加重；3）DDRGK1与RPS3、IRE1a、QSOX1结合；4）DDRGK1敲除，影响RPS3，IRE1a的表达和功能，影响QSOX1的二硫化反应。据此结论：DDRGK1蛋白表达下调或缺失，影响RPS3，IRE1a的类泛素化水平，可能由泛素化系统参与介导蛋白降解。同时，会影响QSOX1酶活，细胞内外稳态失衡，使髓核和软骨退变，加剧椎间盘退变疾病的发生。进一步，我们申请并获批DDRGK1蛋白治疗骨与关节退变的多项专利，目前进入实质性的转化启动阶段，为临床治疗该类疾病提供新策略。